In FY2008, we continued our investigation of the Na+, K+-ATPase (NKA, sodium pump) as an interacting protein for the D1 and D2 receptors. It is well documented that dopamine can increase or decrease the activity of the NKA in an organ-specific fashion. This regulation can occur, at least partially, via receptor-mediated second messenger activation, and can promote NKA insertion or removal from the plasma membrane. Using co-immunoprecipitation and mass spectrometry we show that, in both brain and HEK293T cells, D1 and D2 dopamine receptors (DARs) can exist in a complex with the sodium pump. To determine the impact of NKA on DAR function, biological assays were conducted with NKA and DARs expressed in HEK293T cells. In this system, expression of NKA dramatically decreased D1 and D2 DAR numbers with a concomitant functional decrease in DAR-mediated regulation of cAMP levels. Interestingly, pharmacological inhibition of endogenous or over-expressed NKA enhanced DAR function without altering receptor number or localization. Similarly, DAR function was also augmented by siRNA reduction of the endogenous NKA. These data suggest that, under basal conditions, NKA negatively regulates DAR function via protein-protein interactions. In reciprocal fashion, over-expression of DARs decreases endogenous NKA function in the absence of dopamine, implicating DAR proteins as regulators of NKA activity. Dopamine stimulation or pertussis toxin inhibition of D2 receptor signaling had no impact on NKA activity, indicating that the D2-mediated decrease in NKA function occurs through direct interaction with the receptor protein. These data suggest that DARs and NKA can reciprocally regulate one another via protein-protein interactions, thus providing a control mechanism for both DAR signaling and cellular ion balance.[unreadable] [unreadable] Our laboratory and others have previously shown that the D2 dopamine receptor (DAR) is internalized by agonist stimulation and either recycled back to the plasma membrane or sorted to lysosomes for degradation. However, the molecular components involved in these processes are only beginning to be characterized. In FY2008, we have identified sorting nexin-25 (SNX25) as a novel DAR interacting protein using co-immunoprecipitation-coupled mass spectrometry-based sequencing. Mammalian SNXs have been suggested to be involved in the internalization, intracellular trafficking, and endosomal recycling or sorting of membrane-bound cargo. In addition, hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) has been found to play a distinct role in promoting the rapid recycling of internalized receptors back to the plasma membrane. In FY2008, we investigated the role of SNX25 and Hrs on D2 DAR internalization and recycling in HEK293T cells. Receptor internalization and recycling was initially assessed by quantifying cell surface receptors using intact cell 3H-sulpiride binding assays. Treatment with dopamine for 1 hr resulted in a 25% loss of cell surface receptor binding (internalization), which recovered to 93% of control after 1hr of dopamine removal (recycling). Over-expression of either SNX25 or Hrs had no effect on the total surface receptor expression, but decreased the amount of agonist-induced receptor internalization to 17% and 11%, respectively. Surprisingly, D2 DAR recycling was severely impaired in Hrs over-expressing cells. However, SNX25 over-expression resulted in only a slight decrease in receptor recycling. Taken together, over-expression of either SNX25 or Hrs perturbed both internalization and recycling of the D2 DAR. These data suggest that SNX25 and Hrs may play a role in D2 DAR trafficking through membrane compartments and that common machinery exists linking receptor internalization (endocytosis) and recycling. We next examined the intracellular distribution of D2 DAR-YFP or -RFP constructs in the presence of Hrs over-expression using either myc- or GFP-tagged Hrs. In Hrs over-expressing cells, a significant number of D2 DARs were localized to intracellular regions and were co-localized with Hrs. Furthermore, co-immunoprecipitation experiments revealed that Hrs directly interacts with the D2 DAR. These data suggest that SNX25 and Hrs directly interact with D2 DARs and modulate receptor trafficking when independently over-expressed. Further characterization of SNX25- and Hrs-dependent trafficking pathways and their relationship to each other are currently under investigation.[unreadable] [unreadable] In FY 2008, we also continued our investigations of how protein kinase C regulation of the D1 receptor is regulated by alchohol. Alcohol abuse and alcoholism are of clinical and economic significance worldwide. The effectiveness of the current pharmacotherapies for alcoholism is limited and is partially due to lack of mechanistic data at the molecular level. Aberrant protein kinase C (PKC) signaling is associated with many diseases that include alcoholism and addiction. The PKC family of serine/threonine kinases is comprised of 12 isozymes that differ with respect to their structure, expression, and mechanisms of regulation. In many instances, only a subset of the PKC isozymes is associated with a specific disease state. We are particularly interested in the interplay between PKC and the D1 dopamine receptor in neuropsychiatric disorders such as alcohol abuse and alcoholism. We've recently found that PKC constitutively phosphorylates the D1 receptor and that this negatively regulates dopaminergic signaling. Moreover, we've shown that ethanol (EtOH) treatment decreases constitutive PKC phosphorylation of the D1 receptor with a concomitant potentiation of dopaminergic signaling. Importantly, EtOH was found to directly inhibit the lipid-activated enzymatic activities of PKC&#947; and PKC&#948;, but only when they were isolated from the membrane fraction - a response that was not observed for other PKC isozymes, including &#945;, &#946;1 or &#949;. The molecular mechanisms underlying the EtOH-mediated inhibition of membrane-associated PKC&#947; and PKC&#948; kinase activities are at present unclear. We hypothesize that EtOH may regulate the interaction between the PKC isozyme and a membrane-associated interacting protein(s) and/or target the PKC interacting protein itself. We have mow employed a PKC isozyme-specific coimmunoprecipitation approach followed by 2-D gel electrophoresis and mass spectrometry-based sequencing to identify candidate PKC isozyme-specific interacting proteins. RanBP10, a known scaffolding protein, was identified in the proteomics screen involving PKC&#947;. Significantly, we confirmed the association of RanBP10 with both PKC&#947; and PKC&#948; using coimmunoprecipitation techniques followed by Western Blotting. Since PKC&#947; and PKC&#948; both phosphorylate the D1 receptor, we postulate that RanBP10 may function as a scaffolding molecule and may also associate with the D1 receptor. This notion was supported by the coimmunoprecipitation of RanBP10 with the D1 receptor. Interestingly, our preliminary functional data suggest that RanBP10 modulates D1 receptor expression and cAMP accumulation. In summary, we have identified a putative scaffolding molecule, RanBP10 that may be a critical component that regulates crosstalk between D1 receptor and PKC isozyme-specific signaling pathways.